Estrogenic and antiestrogenic compounds that bind in the estrogen receptor (ER) ligand binding pocket have played key roles in developing our understanding of ER action. The antiestrogenic compound, tamoxifen (Tam), is used extensively to treat breast cancer, but its long-term use is limited because tumors eventually develop tamoxifen resistance. We developed a high throughput screening strategy to identify small molecules that target ER action at the level of ER binding to its DNA response element, rather than the traditional approach of targeting antagonism of estrogen binding. Our lead ER inhibitor, TPSF, potently and specifically inhibits estrogen-ER-mediated gene expression and estrogen-ER-dependent growth of Tam-sensitive MCF-7 cells and three Tam-resistant breast cancer cell lines. However, TPSF has no effect on growth of ER negative cells. The three Specific Aims of this proposal build on our recent identification of TPSF. (Aim 1) Optimize TPSF by using structure-activity relationships to guide synthesis of new compounds for screening and assess inhibitor efficacy, specificity and intracellular actions in cell-based assays using Tam-sensitive and Tam-resistant breast cancer cells. (Aim 2) Identify the site on ER1 that interacts with the inhibitors and their mode of action, and (Aim 3) Test TPSF, and the best inhibitor to emerge from optimization, in mouse xenograft models of Tam-sensitive and Tam-resistant breast cancer. Assays and methods: (Aim 1) We will test the efficacy, potency and specificity of each inhibitor in gene expression assays using ER, AR and GR and examine inhibition of ER actions that do not require direct binding of ER to DNA. Using Tam-sensitive and Tam-resistant breast cancer cells, we will evaluate the inhibitor's ability to alter endogenous gene expression, anchorage-dependent and anchorage-independent cell growth and to re-sensitize breast cancer cells to killing by immune cells. We will test for extranuclear effects on the ERK pathway and other signal transduction pathways and for toxicity in long-term studies. To analyze the lead inhibitor's effects on gene expression patterns, we will perform microarray analysis in ER positive breast cancer cells and in non-tumorigenic breast cells. (Aim 2) To evaluate inhibitor-ER interaction in vitro and in cells, we will use ER mutants containing large ER domains and then do studies with smaller mutations. Photoaffinity labeling, NMR comparison of chemical shifts of free ER and ER-inhibitor complexes, and isothermal titration calorimetry will be used to assess direct interaction of the inhibitors with ER. If feasible, structural studies of ER domain-inhibitor complexes will be performed. We will evaluate potential inhibitor effects on ER dimerization, degradation, phosphorylation and synergy with known antagonists. (Aim 3) Using Tam-sensitive and Tam-resistant breast cancer cells, we will assess the ability of the inhibitors to block tumor growth or induce tumor regression. These small molecule inhibitors are powerful new probes for ER action in breast cancer. PUBLIC HEALTH RELEVANCE: Breast cancers that depend on estrogens bound to estrogen receptor for their growth eventually become resistant to drugs that target estrogen. We have identified new inhibitors that bypasses the site targeted by current drugs and instead target an essential action of the estrogen receptor. Further development of these inhibitors may lead to clinically useful drugs effective against tumors that are resistant to current therapies.